Compounds and Methods for the Treatment of Vascular Disease

ABSTRACT

The present invention relates to a method for the treatment of vascular dysfunction, reducing ischemic pain and/or treatment of a vascular disease comprising administering a therapeutically effective amount of Annexin A5 or a functional analogue or variant thereof to a patient in need of such treatment. The vascular dysfunction, ischemic pain and/or vascular disease may be associated with impaired endothelium mediated vasodilatation, a reduced eNOS activity, and/or a reduced NO bioavailability. The patient may be suffering from a disease selected from angina pectoris, ischemic heart disease, peripheral artery disease, systolic hypertension, migraine, type 2 diabetes and erectile dysfunction.

FIELD OF THE INVENTION

The invention relates to novel methods and compositions for thetreatment of vascular dysfunction in patients, such as those sufferingfrom conditions including angina pectoris, ischaemic heart disease,peripheral artery disease, systolic hypertension, migraine, type 2diabetes and erectile dysfunction.

BACKGROUND TO THE INVENTION

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

Angina pectoris (‘tight chest’) is chest pain due to lack of oxygen inthe myocardium. The lack of oxygen is normally due to ischaemia, areduction in blood supply (and, hence, oxygen supply) to the myocardium.Normally this ischaemia is caused by atherosclerotic plaques in thecoronary vessels, but it can also be caused by a local spasm in thecoronary vasculature. Other rare causes can be valve disease, severeanemia, aortic stenoses and tachyarrythmias. Angina pectoris is dividedinto effort angina, angina triggered by physical and/or mental exertion;spasm-angina (variant or Prinzmetal's angina), sudden angina withoutcorrelation to a specific situation; and syndrome X, typically an effortrelated angina but without overt stenosis on angiography. An anginaattack normally has a duration of 1 to 5 minutes. If attacks occur atrest, or have a duration exceeding 15 minutes, the disease is referredto as unstable angina and is associated to imminent risk of acardiovascular event. The condition is classified as unstable anginaalso when stable angina symptoms are worsened. Unstable angina belongsto the Acute Coronary Syndrome (ACS) and is a critical condition. Amyocardial infarction is normally caused by almost complete absence ofblood supply to a part of the myocardium, normally caused by the ruptureof a coronary atherosclerotic lesion with a subsequent formation of anoccluding thrombus.

Peripheral arterial disease (PAD) is a condition with similarities toangina pectoris, but is present in peripheral arteries, normally thelower extremities. Ischaemic pain is common in these patients, and isalso normally associated to physical activity. The pain, or crampingsensation, often experienced as a result of physical exercise in PADpatients is normally referred to as intermittent claudication.

Endothelium in Cardiovascular (CV) disease. Vascular dysfunction, in ageneral sense, characterises most CV disease states, and often involvesaltered endothelial function. The endothelium is the innermost celllayer in all blood vessels. It is the body's largest endocrine gland,and secretes a number of important factors controlling the circulatorysystem. Several recent studies have shown that ‘endothelial dysfunction’is related to an increased risk for CV events [1]. ‘Endothelialdysfunction’ is normally measured as a loss of endothelium mediateddilatation, the capacity the endothelium has to dilate blood vessels inresponse to certain stimuli [2]. There are several ways to measureendothelium mediated dilatation, the most common is dilation of thebrachial artery during hyperemia, flow mediated vasodilatation (FMD)[3]. Using such measurements of endothelium mediated dilatation it isshown that vascular function is hampered in individuals suffering fromatherosclerosis related diseases (hypertension, hyperlipidemia,diabetes) [4, 5].

The release of Nitric Oxide, NO, from the endothelium is a key event inendothelium mediated dilatation [6]. The key enzyme in the generation ofNO is endothelial nitric oxide synthase, eNOS. It has been mostlystudied in direct conjunction to the regulation of the vascular system(thrombosis/haemostasis, blood flow regulation and blood vessel growth),but it is also related to the development of atherosclerosis as well asinsulin resistance and/or type 2 diabetes (T2DM). For example, micedeficient in eNOS are more prone to become atherosclerotic than micewith normal eNOS function and are also insulin resistant [7].

Normalisation of vascular (dys-)function in an arterial disease state,measured as restoration of normal endothelium mediated dilatation, mayor may not be the result of increased release of NO from theendothelium. Vascular dysfunction may, or may not, result from reducedsensitivity of arterial smooth muscles to the NO and/or may, or may not,result from increased metabolism of the NO that is generated. Othermechanisms can also alter vascular function in arterial disease; forexample, it is well known that during vascular inflammation theformation of pro-inflammatory and vasoconstrictor substances areincreased, and this could offset vasodilatory effects, such as thosecaused by NO.

Endothelium in angina pectoris and peripheral arterial disease. Whenacetylcholine is administered into coronary arteries, it triggers therelease of NO from the coronary endothelium, which in turn causesdilatation of the coronaries (endothelium mediated dilatation). Whenthis procedure was performed in patients with coronary artery disease,they responded with a ‘paradoxical vasoconstriction’ [8]. Since then, ithas been shown that there is an inappropriate loss of endotheliummediated dilatation in patients suffering from any form of anginapectoris [9, 10], and that this altered function is an importantcontributor to myocardial ischaemia and, hence, angina pain in thesepatients.

PAD (normally measured as the reduction in ankle-brachial index (ABI)due to stenosing atherosclerotic lesions) is also characterized by areduction in endothelium mediated dilatation [11]. In patients where thedisease is symptomatic (intermittent claudication) the reduction inendothelium mediated dilatation is greater than in non-symptomaticpatients [12].

Systolic hypertension is an important disease in ageing populations, andis normally associated to increased stiffness of the central arterialcompartment [13]. It is also shown that NO reduces stiffness [14], andan agent that can restore reduced endothelium mediated dilatation tonormal in a patient with systolic hypertension can thus be oftherapeutic value.

Another interesting observation is that patients suffering from migrainealso have impaired endothelium mediated dilatation, and it was suggestedthat vascular vasomotion abnormalities can be an importantpathophysiological factor in migraine [15, 16]. It is in this contextinteresting that a mutation in the eNOS gene that is associated to lossof function [17] and is associated to increased risk for CV events[18-20], is also associated to migraine [21].

In yet another aspect of the role of endothelium and NO in disease,erectile dysfunction is also associated with impaired endotheliummediated dilatation [22], and restoring normal endothelium mediateddilatation may also improve erectile dysfunction.

From the above summary, it becomes evident that restoring normalendothelial control of vascular tone by normalising endothelial NOmetabolism is a therapeutic opportunity in disease conditions whereendothelium mediated dilatation is reduced.

As described above, endothelium mediated dilatation is an importantfactor in the development of arterial disease. It is also well knownthat these diseases are also linked to alterations in the haemostaticsystem, and that vascular inflammation is associated to a state wherethere is increased risk for arterial thrombus formation.

Annexin A5 is an endogenous protein that binds to charged phospholipidssuch as phosphatidylserine (PS) [23]. Annexin A5 is a potentanti-thrombotic agent [24], and it is proposed that Annexin A5 bybinding to exposed PS can form a ‘protective shield’ that can inhibitthe effects of PS on thrombosis formation [25]. It is interesting inthis context that in patients with autoimmune disorders such as APSand/or SLE, there are antibodies in the plasma that can reduce thebinding of Annexin A5 to PS on e.g. endothelial cell surfaces, therebyincreasing PS exposure. This finding may explain why these patients areat a higher risk for thrombotic events than the general population [23].Very interestingly, there was a significant reduction in Annexin A5binding capacity to endothelial cells of serum from controls to SLEpatients that had not suffered thrombotic events to SLE patients thathad suffered such events [26].

It has been shown that in addition to anti-platelet and anti-coagulanteffects of Annexin A5, this protein and an analogue thereof, the AnnexinA5 dimer diannexin, is effective in preventing against reperfusioninjury in the liver [27], and it improved the outcome of rat livertransplants [28]. Interestingly, in both these studies the treatmentswere associated with a reduced inflammatory activity in the hepaticendothelium, measured as reduced expression of adhesion molecules. Itwas suggested that diannexin improved the survival of the livertransplants by an anti-thrombotic effect leading to maintained bloodsupply to the liver [28].

It has earlier been suggested that Annexin A5 can be used to stabiliseatherosclerotic lesions in coronary arteries in patients, which shouldreduce the risk for myocardial infarction in these patients ([26]; WO2005/099744).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Annexin A5 restore endothelium mediated vasodilatation

Representative recordings of blood pressure in a control apoE^((−/−))mouse (upper trace) and an Annexin A5 treated mouse (lower trace).Metacholine was injected as indicated by the dotted line. Panels to theright with previous administration of the inhibitor of eNOS, 1-NAME.

FIG. 2. Summary of the effects of Annexin A5 on endothelium mediatedvasodilatation measured as blood pressure.

The bars show mean arterial pressure before (basal) and 3 minutes afterthe administration of metacholine in untreated and Annexin A5 treatedmice, respectively.

FIG. 3. Summary of the effects of Annexin A5 on metacholine inducedchanges in systolic and diastolic blood pressure.

Metacholine was injected at time 0 in either control (-♦-) or Annexin A5treated atherosclerotic mice Effect on systolic (•••▪•••) and diastolic(B) blood pressure is shown.

DESCRIPTION OF THE INVENTION

Here we show that Annexin A5 can restore normal endothelium mediatedvasodilatation in atherosclerotic mice, by a NO dependent mechanism.Thus, Annexin A5 or analogues thereof provide a new treatment modalitywith anti-ischaemic effects that can normalise vasomotion abnormalitiesin diseases where this abnormality is due to a hampered eNOS function orNO bioavailability. There are no previous reports to show that AnnexinA5 has anti-ischaemic properties. Such diseases include, but are notlimited to, angina pectoris, ischaemic heart disease, peripheral arterydisease, systolic hypertension, migraine, type 2 diabetes and erectiledysfunction.

In a first aspect the invention provides a method for the treatment ofvascular dysfunction and/or for restoring vascular function comprisingadministering a therapeutically effective amount of Annexin A5 or afunctional analogue or variant thereof to a patient in need of suchtreatment.

Put another way, the first aspect of the present invention providesAnnexin A5 or a functional analogue or variant thereof for use in thetreatment of vascular dysfunction and/or for restoring vascularfunction.

In one embodiment, a patient may be said to suffer from vasculardysfunction if the patient displays a reduced ability to respond to avasodilating stimulus (optionally, when the mean average response of thepatient is assessed over multiple, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10,repetitions of the test) compared to the mean average ability of acontrol group (10 healthy age-matched, and sex-matched, individuals,optionally wherein each member of the control group is assessed overmultiple, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, repetitions of the test)to respond to the same dosage of the vasodilating stimulus. Suitablevasodilating stimuli that may be used in this assessment include stimuliselected from the group consisting of metacholine, acetylcholine,increased blood flow rate, an NO-generating compound such asnitroglycerine, or a functional test such as an exercise test frequentlyused in angina patients.

The ability of an individual to respond to a vasodilating stimulus canbe assessed by measuring the change in vasodilation caused by theadministration of the vasodilating stimulus by intravenous,intra-arterial, sublingual, oral or subcutaneous administration (wherethe vasodilating stimulus is an agent to be administered to the testsubject) or by measuring the response to a functional test, such as anexercise test.

Methods to measure changes in vasodilation are known in the art, and themost appropriate method will be chosen depending on the nature of thevascular dysfunction and/or the type of vasodilating stimulus used.

A common way to measure vasodilation in a test subject, for the purposesof assessing vascular dysfunction, is to measure flow mediateddilatation of the brachial artery following ischaemia induced by a cuffon the upper arm [3]. Previous reports of using this technique haveshown that, typically, post-ischaemic hyperemia causes an increase inbrachial artery diameter by approximately 10% in the young and healthyindividual, whereas in a CVD patient or a type 2 diabetic patient, thisresponse may be e.g. 2-4% or even completely absent. The response can bealmost completely inhibited by the administration of an inhibitor of theNO generating enzyme such as 1-NAME.

Alternatively, vasodilation in the resistance arteries of a test subject(e.g. arteries in the lower arm and/or hand) can be measured as changesin blood flow using the art-known technique of plethysmography. In thisexperimental setting, the vasodilating stimulus could optionally be alocal (such as, an intra-arterial) administration of a vasodilatingsubstance, such as acetylcholine.

Coronary vascular function can be assessed by an exercise test such as abicycle exercise test or a treadmill walk test. In this situation,cardiac ischaemia is measured by changes in the cardiacelectrocardiogram (ECG), and/or as the amount of physical work a subjectcan perform before ischaemic pain (angina) occurs.

Alternatively, large coronary artery function can be evaluated by thelocal injection of a vasodilating compound during cardiaccatheterisation, and the response is evaluated by coronary angiography.

Thus, a patient suffering from vascular dysfunction may display anability to respond to a vasodilating stimulus that is, at most, 95%,90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less than the to themean average ability of the control group to respond to the same dosageof the vasodilating stimulus.

Patients suffering from diseases such as angina pectoris, ischaemicheart disease, peripheral artery disease, systolic hypertension,migraine, type 2 diabetes and erectile dysfunction commonly displayvascular dysfunction.

Thus, vascular dysfunction may be treated and/or vascular functionrestored by administering a therapeutically effective amount of AnnexinA5 or a functional analogue or variant thereof to a patient in needthereof. A therapeutically effective amount of Annexin A5 or afunctional analogue or variant thereof, when used in a method accordingto the present invention to treat vascular dysfunction and/or restorevascular function, can be assessed either by—

-   (i) monitoring the response, to a vasodilating stimulus, of a    patient suffering from vascular dysfunction, and comparing the    observed response before, and then after treatment with Annexin A5    or a functional analogue or variant thereof; and/or-   (ii) monitoring the response, to a vasodilating stimulus, of a    patient suffering from vascular dysfunction that has been treated    with Annexin A5 or a functional analogue or variant thereof, and    comparing the observed response to the mean average ability of the    control group of healthy individuals (as defined above, wherein the    members of the control group have not been treated with Annexin A5    or a functional analogue or variant thereof) to respond to the same    dosage of the vasodilating stimulus; and/or-   (iii) monitoring the response, to a vasodilating stimulus, of a    patient suffering from vascular dysfunction that has been treated    with Annexin A5 or a functional analogue or variant thereof, and    comparing the observed response to the mean average ability of a    control group of condition-matched individuals (10    condition-matched, age-matched, and sex-matched, individuals,    optionally wherein each member of the control group is assessed over    multiple, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, repetitions of the    test), wherein the members of the control group have not been    treated with Annexin A5 or a functional analogue or variant thereof,    to respond to the same dosage of the vasodilating stimulus.

The ability of an individual to respond to a vasodilating stimulus canbe assessed by measuring the change in vasodilation caused by theadministration of the vasodilating stimulus as set out above.

Where the therapeutic effect of the Annexin A5 or a functional analogueor variant thereof is determined as in (i) above, then the level ofvasodilating stimulus-induced vasodilatation achieved in the patientprior to treatment with Annexin A5 or a functional analogue or variantthereof may be improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95% or at least 100% or even higher values, such as atleast 150%, 200%, 250%, 300% or more, by treatment with atherapeutically effective amount of Annexin A5 or a functional analogueor variant thereof in accordance with the present invention, compared tothe pre-treatment level of vasodilating stimulus-induced vasodilatationachieved in the patient.

Where the therapeutic effect of the Annexin A5 or a functional analogueor variant thereof is determined as in (ii) above, then the level ofvasodilating stimulus-induced vasodilatation achieved in the patientfollowing treatment with Annexin A5 or a functional analogue or variantthereof may be improved to a level that is at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 85% 90%, 95%, 98%, 99%, substantially about 100% ormore compared to the mean average level of vasodilating stimulus-inducedvasodilation observed in the control group.

Where the therapeutic effect of the Annexin A5 or a functional analogueor variant thereof is determined as in (iii) above, then the level ofvasodilating stimulus-induced vasodilatation achieved in the patientfollowing treatment with Annexin A5 or a functional analogue or variantthereof may be improved to a level that is at greater than 100%, such asat least 150%, 200%, 250%, 300%, or more, compared to the mean averagelevel of vasodilating stimulus-induced vasodilation observed in thecontrol group.

The vascular dysfunction may be associated with impaired endotheliummediated vasodilatation and/or may be associated with a reduced eNOSactivity and/or a reduced NO bioavailability.

The present invention also provides, as a second aspect, a method forreducing ischemic pain, such as angina pectoris or pain associated withperipheral arterial disease (PAD), by administering a therapeuticallyeffective amount of Annexin A5 or a functional analogue or variantthereof to a patient in need of such treatment.

Put another way, the second aspect of the present invention providesAnnexin A5 or a functional analogue or variant thereof for use inreducing ischemic pain.

The patient treated by the first or second aspect of the invention may,or may not, be a subject suffering from a disease selected from anginapectoris, ischaemic heart disease, peripheral artery disease, systolichypertension, migraine, type 2 diabetes and erectile dysfunction, andthus the vascular dysfunction and/or ischemic pain may be associatedwith any one or more of these diseases.

Where the patient is suffering from angina pectoris, it may, or may not,be caused by any one of atherosclerotic plaques in the coronary vessels,local spasm in the coronary vasculature, valve disease, severe anaemia,aortic stenoses, and/or tachyarrythmias. The patient may, or may not, besuffering from effort angina, spasm-angina (variant or Prinzmetal'sangina), sudden angina, or syndrome X. The patient may, or may not, besuffering from stable, or unstable, angina.

Treatment of vascular dysfunction and/or restoring vascular function inpatients with conditions such as angina pectoris in accordance with thepresent invention may also reduce the risk of the onset of acutemyocardial infarction (AMI) and its following complications such asthose listed by the international classification of disease (ICD) athttp://www.who.int/classifications/apps/icd/icd10online/, under categoryI.23 which includes haemopericardium following AMI (I23.0), atrialseptal following AMI (I23.1), ventricular septal defect following AMI(I23.2), rupture of cardiac wall without haemopericardium following AMI(I23.3), rupture of chordae tendineae following AMI (I23.4), rupture ofpapillary muscle following AMI (I23.5), thrombosis of atrium, auricularappendage, and ventricle following AMI (I23.6) and other currentcomplications following AMI (I23.8).

Where the patient is suffering from ischemic heart disease, the patientmay, or may not, suffer from atherosclerosis, or angina without overtatherosclerosis.

The patient may, or may not, be a human. The patient may, or may not bea non-human animal, such as a domestic animal (for example, cat, dog,rabbit, cow, sheep, pig, mouse or other rodent).

In a third aspect, the invention provides a method for the treatment,prevention, or reduction of risk of onset of a vascular diseasecomprising administering a therapeutically effective amount of AnnexinA5 or a functional analogue or variant thereof to a patient in need ofsuch treatment.

Put another way, the third aspect of the present invention providesAnnexin A5 or a functional analogue or variant thereof for use in thetreatment, prevention, or reduction of risk of onset of a vasculardisease.

The vascular disease treated by the third aspect of the invention can beassociated with impaired endothelium mediated vasodilatation, reducedeNOS activity and/or reduced NO bioavailability. The vascular diseasetreated by the third aspect of the invention may, or may not, be adisease selected from angina pectoris, ischaemic heart disease,peripheral artery disease, systolic hypertension, migraine, type 2diabetes and erectile dysfunction, such as those conditions discussedabove in more detail in respect of the first and second aspects of theinvention.

Treatment of conditions such as angina pectoris in accordance with thepresent invention may also reduce the risk of the onset of acutemyocardial infarction (AMI) and its following complications such asthose listed by the international classification of disease (ICD) athttp://www.who.int/classifications/apps/icd/icd10online/, under categoryI.23 which includes haemopericardium following AMI (I23.0), atrialseptal following AMI (I23.1), ventricular septal defect following AMI(I23.2), rupture of cardiac wall without haemopericardium following AMI(I23.3), rupture of chordae tendineae following AMI (I23.4), rupture ofpapillary muscle following AMI (I23.5), thrombosis of atrium, auricularappendage, and ventricle following AMI (I23.6) and other currentcomplications following AMI (I23.8).

Thus, the third aspect of the present invention includes a method forreducing the risk of onset of AMI or its following complications by thetreatment of a vascular disease comprising administering atherapeutically effective amount of Annexin A5 or a functional analogueor variant thereof to a patient.

The Annexin A5 or the functional analogue or variant thereof can beadministered in conjunction with (that is, separately, simultaneously,or sequentially with) one or more further active agent(s), such as—

-   -   a thrombolytic therapeutic, such as tissue plasminogen        activator, urokinase, or a bacterial enzyme,    -   an antiplatelet agent, such as aspirin or clopidogrel (Plavix),        and/or    -   nitroglycerin and/or morphine.

In a fourth aspect, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of Annexin A5 or afunctional analogue or variant thereof for the treatment of a vasculardisease selected from angina pectoris, ischaemic heart disease,peripheral artery disease, systolic hypertension, migraine, and erectiledysfunction, as discussed above in respect of the first and secondaspect of the invention. The vascular disease can be associated with (inother words, a patient with the vascular disease may display) impairedendothelium mediated vasodilatation, a reduced eNOS activity and/or areduced NO bioavailability.

In a fifth aspect, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of Annexin A5 or afunctional analogue or variant thereof for the treatment of a vasculardysfunction and/or restoring vascular function. The vascular dysfunctioncan be associated with (in other words, a patient with the vasculardysfunction may display) impaired endothelium mediated vasodilatation, areduced eNOS activity, and/or a reduced NO bioavailability. The vasculardysfunction can be associated with a disease selected from anginapectoris, ischaemic heart disease, peripheral artery disease, systolichypertension, migraine, type 2 diabetes and erectile dysfunction, asdiscussed above in respect of the first and second aspect of theinvention.

A pharmaceutical composition according to the fourth or fifth aspects ofthe invention may thus comprise Annexin A5 or a functional analogue orvariant thereof in admixture with a pharmaceutically or veterinarilyacceptable adjuvant, diluent or carrier, which will typically beselected with regard to the intended route of administration andstandard pharmaceutical practice. The composition may be in the form ofimmediate-, delayed- or controlled-release applications. Preferably, theformulation is a unit dosage containing a daily dose or unit, dailysub-dose or an appropriate fraction thereof, of the active ingredient.

The pharmaceutical composition according to the invention may, or maynot, be intended for, and, thus formulated in a manner suitable for,parenteral, intravenous, intra-arterial, intraperitoneal, intra-muscularor subcutaneous administration, or they may be administered by infusiontechniques. They may be best used in the form of a sterile aqueoussolution which may contain other substances, for example, enough saltsor glucose to make the solution isotonic with blood. The aqueoussolutions may be suitably buffered (preferably to a pH of from 3 to 9),if necessary. The preparation of suitable pharmaceutical formulationsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well-known to those skilled in the art.

Such formulations may include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampoules and vials, and may be stored in a freeze-dried(lyophilised) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed.

A therapeutically effective amount of Annexin A5 or a functionalanalogue or variant thereof for administration to a patient, such as ahuman patient, on the basis of a daily dosage level may be from 0.01 to1000 mg of Annexin A5 or a functional analogue or variant thereof peradult (for example, from about 0.001 to 20 mg per kg of the patient'sbody weight, such as 0.01 to 10 mg/kg, for example greater than 0.1mg/kg and less than 20, 10, 5, 4, 3 or 2 mg/kg, such as about 1 mg/kg),administered in single or divided doses.

The physician in any event will determine the actual dosage which willbe most suitable for any individual patient and it will vary with theage, weight and response of the particular patient. The above dosagesare exemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited and such arewithin the scope of this invention.

For veterinary use, a compound of the invention is administered as asuitably acceptable formulation in accordance with normal veterinarypractice and the veterinary surgeon will determine the dosing regimenand route of administration which will be most appropriate for aparticular animal.

In a sixth aspect, the invention provides use of Annexin A5 or afunctional analogue or variant thereof in the manufacture of a medicinalproduct for the treatment, prevention or reduction of risk of onset of acondition defined above in the context of any one of the first, secondor third aspects of the present invention.

The Annexin A5 or the functional analogue or variant thereof accordingto the invention can be human Annexin A5 (SEQ ID NO:1), an allelic orgenetic variant thereof, a mammalian orthologue thereof, or an allelicor genetic variant thereof. Preferably the functional analogue orvariant of Annexin A5 according to the invention is more than 50%, 60%,70%, 75%, such as more than 80% or 85%, more than 90%, or preferablymore than 95% or 99% identical to human Annexin A5, SEQ ID NO:1.

The percent identity between two amino acid sequences is determined asfollows. First, an amino acid sequence is compared to, for example, SEQID NO:1 using the BLAST 2 Sequences (Bl2seq) program from thestand-alone version of BLASTZ containing BLASTN version 2.0.14 andBLASTP version 2.0.14. This stand-alone version of BLASTZ can beobtained from the U.S. government's National Center for BiotechnologyInformation web site at ncbi.nlm.nih.gov. Instructions explaining how touse the Bl2seq program can be found in the readme file accompanyingBLASTZ. Bl2seq performs a comparison between two amino acid sequencesusing the BLASTP algorithm. To compare two amino acid sequences, theoptions of Bl2seq are set as follows: -i is set to a file containing thefirst amino acid sequence to be compared (e.g., C:\seq1.txt); -j is setto a file containing the second amino acid sequence to be compared(e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired filename (e.g., C:\output.txt); and all other options are left at theirdefault setting. For example, the following command can be used togenerate an output file containing a comparison between two amino acidsequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -oc:\output.txt. If the two compared sequences share homology, then thedesignated output file will present those regions of homology as alignedsequences. If the two compared sequences do not share homology, then thedesignated output file will not present aligned sequences. Once aligned,the number of matches is determined by counting the number of positionswhere an identical nucleotide or amino acid residue is presented in bothsequences.

The percent identity is determined by dividing the number of matches bythe length of the sequence set forth in an identified sequence followedby multiplying the resulting value by 100. For example, if a sequence iscompared to the sequence set forth in SEQ ID NO:1 (the length of thesequence set forth in SEQ ID NO:1 is 320) and the number of matches is288, then the sequence has a percent identity of 90 (i.e., 288320*100=90) to the sequence set forth in SEQ ID NO:1.

Thus, a functional analogue or variant of Annexin A5 may be a proteinwherein at one or more positions there have been amino acid insertions,deletions, or substitutions, either conservative or non-conservative,provided that such changes result in a protein whose basic properties tofunction in an equivalent manner to Annexin A5 have not significantlybeen changed. “Significantly” in this context means that one skilled inthe art would say that the properties of the variant may still bedifferent but would not be unobvious over the ones of the originalprotein.

By “conservative substitutions” is intended combinations such as Gly,Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe,Tyr.

Such variants may be made using the methods of protein engineering andsite-directed mutagenesis which are well known in the art.

The functional analogue or variant of Annexin A5 according to theinvention may, or may not, be a dimer of Annexin A5 or a functionalanalogue or variant thereof, or may or may not, be a PEGylated AnnexinA5 or a functional analogue or variant thereof. DiAnnexinA5 andPEGylated AnnexinA5 are disclosed in WO 02/067857.

PEGylation is a method well known to those skilled in the art wherein apolypeptide or peptidomimetic compound (for the purposes of the presentinvention, Annexin A5 or the functional analogue or variant) is modifiedsuch that one or more polyethylene glycol (PEG) molecules are covalentlyattached to the side chain of one or more amino acids or derivativesthereof. It is one of the most important molecule altering structuralchemistry techniques (MASC). Other MASC techniques may be used; suchtechniques may improve the pharmacodynamic properties of the molecule,for example extending its half life in vivo. A PEG-protein conjugate isformed by first activating the PEG moiety so that it will react with,and couple to, the protein or peptidomimetic compound of the invention.PEG moieties vary considerably in molecular weight and conformation,with the early moieties (monofunctional PEGs; mPEGs) being linear withmolecular weights of 12 kDa or less, and later moieties being ofincreased molecular weights. PEG2, a recent innovation in PEGtechnology, involves the coupling of a 30 kDa (or less) mPEG to a lysineamino acid (although PEGylation can be extended to the addition of PEGto other amino acids) that is further reacted to form a branchedstructure that behaves like a linear mPEG of much greater molecularweight (Kozlowski et al., (2001), Biodrugs 15, 419-429). Methods thatmay be used to covalently attach the PEG molecules to polypeptides arefurther described in Roberts et al., (2002) Adv Drug Deliv Rev, 54,459-476, Bhadra et al., (2002) Pharmazie 57, 5-29, Kozlowski et al.,(2001) J Control Release 72, 217-224, and Veronese (2001) Biomaterials22, 405-417 and references referred to therein.

The advantages of PEGylation to the polypeptide or peptidomimeticcompound of the invention include reduced renal clearance which, forsome products, results in a more sustained adsorption afteradministration as well as restricted distribution, possibly leading to amore constant and sustained plasma concentrations and hence an increasein clinical effectiveness (Harris et al., (2001) Clin Pharmacokinet 40,539-551). Further advantages can include reduced immunogenicity of thetherapeutic compound (Reddy, (2001) Ann Pharmacother 34, 915-923), andlower toxicity (Kozlowski et al., (2001), Biodrugs 15, 419-429).

The functional analogue or variant of Annexin A5 according to theinvention can be a fusion protein comprising the sequence of Annexin A5or a variant thereof. Thus, for example, Annexin A5 or a variant thereofcan be fused to one or more fusion partner polypeptide sequence(s) so asto extend the half-life of the molecule within a patient's circulatorysystem and/or add further functionality to the molecule.

By a “functional” analogue or variant of Annexin A5 is meant a proteincapable of binding to phosphatidylserine on a biological membrane,preferably to a level that is at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99% or about 100% of that displayed by human AnnexinA5 (SEQ ID NO:1) under the same conditions. A suitable method formeasuring Annexin A5 binding to phosphatidylserine on a biologicalmembrane is known in the art [29].

A “functional” analogue or variant of Annexin A5 may, additionally, oralternatively, also possess at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 99% or about 100% of the therapeutic activity humanAnnexin A5 (SEQ ID NO:1) when used at the same (i.e. molar equivalent)dosage, for treatment of vascular dysfunction, for restoring vascularfunction, for reducing ischemic pain and/or for the treatment of avascular disease in accordance with any of the first, second or thirdaspects of the invention.

In this context, the therapeutic activity of a “functional” analogue orvariant of Annexin A5 may be determined, compared to that of humanAnnexin A5 (SEQ ID NO:1), by comparing the ability of a molar equivalentamount of the functional analogue or variant and of human Annexin A5 totreat vascular dysfunction as described above, and/or to restore theability of apoE^((−/−)) mice kept on a diet of high lipid andcholesterol for 4 months to display endothelium mediated dilation inresponse to intraperitoneal administration of 3 μg/kg bw (10 μl/g)metacholine (Sigma), when the mice are administered, by intraperitonealinjection, molar equivalent amount of either Annexin A5 or the analogueor variant in question once a day in the morning for 3 consecutive days,the final injection being approximately 2-3 hours before theadministration of metacholine, and wherein the extent of endotheliummediated dilation in the test subjects is measured by assessing bloodpressure via catheterisation of the left carotid artery, such as usingthe methodology set out in the following example.

“Vascular disease associated with a reduced eNOS activity or a reducedNO bioavailability” as used herein refers to a vascular disease which iscaused by, causes or leads to, has a common cause with, or isco-existing with a reduced eNOS activity or a reduced NObioavailability.

“Vascular dysfunction associated with a disease” as used herein refersto a vascular dysfunction, such as a loss of endothelium mediatedvasodilatation, which is caused by, causes or leads to, has a commoncause with, or is co-existing with the disease in question. Examples ofsuch diseases are provided above.

EXAMPLE

Mice that are made deficient in apolipoprotein E by gene targeting(apoE^((−/−)) mice) are hyperlipidemic and develop atherosclerosis. Thisprocess is accelerated by feeding the mice a so called Western diet,e.g. Harlan Teklad TD.88137. This is a purified diet with 21% anhydrousmilkfat (butterfat), 34% sucrose, and a total of 0.2% cholesterol which,compared to normal mouse diets, contains extra lipids and somecholesterol. We gave a such diet to male apoE^((−/−)) mice for at least4 months, after which we evaluated the effects of Annexin A5 onendothelium mediated dilatation. In this way, the mice will developvascular disease including an impairment of endothelium mediateddilatation that is typical for patients with e.g. angina pectoris,hypertension, diabetes and hyperlipidemia.

Evaluation of Endothelium Mediated Dilatation in Anaesthetized Mice.

Anaesthesia was induced by 4.5% Isofluran (Isoba®vet, Schering-PloughAnimal Health, Denmark) and then maintained by supplying Isofluran asneeded (normally 1.5-2%).

For blood pressure measurements we performed a catheterisation (SambaSensor preclin 420 LP, Västra Frölunda, Sweden) of the left carotidartery. Thereafter the catheter was gently moved into aortic arch. TheSamba sensor was connected to a Samba 3200 unit, from which the data wastransferred to and stored in the Powerlab software (AD Instrument,version 5) at 2 kHz.

Mice were then allowed to stabilise for approx. 15 minutes. Endotheliummediated dilatation was then induced by the intraperitoneal (ip)administration of 3 μg/kg bw (10 μl/g) metacholine (Sigma), and bloodpressure was measured for 5 minutes. When endothelium mediateddilatation is stimulated systemically, total peripheral vascularresistance is reduced, and the vasodilatation can be measured as areduction in arterial blood pressure. Metacholine causes a transientstimulation of the arterial endothelium to release NO, which in turnwill cause endothelium mediated vasodilatation in a normal, healthymouse. After the effect of metacholine has disappeared and baselinepressure is again established, an ip dose of 50 mg/kg bw L-NAME(N^(G)-nitro-L-arginine methyl ester) (Sigma) was given. This is aninhibitor of the enzyme eNOS, which converts arginine to NO (andcitrulline), and thus responsible for the endothelium mediateddilatation. Blood pressure was then followed until a new plateau wasreached (12-15 minutes), after which metacholine 3 μg/kg was againadministered and blood pressure measured for 5 minutes.

Results.

Normal mice will respond to metacholine injections by a reduction inboth systolic and diastolic blood pressure. After 4 months on thewestern diet, a pilot study of some apoE^((−/−)) mice was performed toverify that the normal response to a metacho line injection was absent.When this had been established, mice received 1 mg recombinant humanAnnexin A5 (Bender Medsystems, Vienna, Austria)/kg bw or its vehicle(saline) in the morning for 3 days by intraperitoneal injection. On the3^(rd) day, and approximately 2-3 hours after the final injection ofAnnexin A5/vehicle, endothelium mediated dilatation was investigated asdescribed above. FIG. 1 is an example of a typical experiment:

FIG. 1 shows representative recordings of blood pressure in a controlapoE^((−/−)) mouse (upper trace) and an Annexin A5 treated mouse (lowertrace). Metacholine was injected as indicated by the dotted line. Themouse treated with Annexin A5 responded with a profound vasodilatation,a marked improvement compared to the control mouse, that hardlyresponded at all (left panels). The panel to the right shows that thisresponse was inhibited by previous administration of the inhibitor ofeNOS, 1-NAME, verifying that the effect of Annexin A5 on endotheliummediated dilatation was mediated by NO. The reduction in systolic bloodpressure was larger than the reduction in diastolic blood pressure.

FIG. 2 summarises the effects of Annexin A5 on endothelium mediateddilatation in this model of angina pectoris and shows that in theuntreated apoE^((−/−)) mice injection of metacholine did not reduce meanblood pressure. In contrast, Annexin A5 treated apoE^((−/−)) miceresponded to metacholine with a significant reduction in blood pressure.As this effect was blocked by 1-NAME we concluded that the effect wasmediated by eNOS/nitric oxide.

FIG. 3 shows that Annexin A5 treatment for 3 days significantly improvedthe endothelium mediated reduction in both systolic (A) and diastolic(B) blood pressures.

Treatment with Annexin A5 restored not only the function of resistancearteries as indicated by the reduction in diastolic blood pressure, butas the effect on systolic blood pressure was larger than effects ondiastolic blood pressure, the drug treatment also increased complianceof the central arteries.

This study shows for the first time that Annexin A5 can restore normalfunction of the endothelium in a mouse model of artery disease/anginapectoris. The effect was mediated by restoration of the NO metabolism inthe endothelium. The findings show the potential of Annexin A5 to exertanti-ischaemic effects. The most common treatment of angina pectoris isrestore blood flow to ischaemic myocardial tissue by the administrationof exogenous NO, normally in the form of organic nitrates such asnitroglycerin. As repeated use of exogenous organic nitrates isassociated to development of nitrate tolerance, this treatment isnormally limited to acute ischaemic situations. Here we have shown thepotential of Annexin A5 as an alternative anti-ischaemic treatment invascular diseases in which normal NO metabolism is hampered.

Summary. Arterial vasomotion abnormalities due to impaired endotheliummediated dilatation is common in most patients with ischaemic vasculardisease. In a model of such disease, it was surprisingly found thatAnnexin A5 restored the impaired endothelium mediated function through aNO dependent mechanism.

REFERENCES

-   1. Lerman, A. and A. M. Zeiher, Endothelial function: cardiac    events. Circulation, 2005. 111(3): p. 363-8.-   2. Landmesser, U., B. Hornig, and H. Drexler, Endothelial function:    a critical determinant in atherosclerosis? Circulation, 2004. 109(21    Suppl 1): p. 1127-33.-   3. Sorensen, K. E., et al., Non-invasive measurement of human    endothelium dependent arterial responses: accuracy and    reproducibility. Br Heart 3, 1995. 74(3): p. 247-53.-   4. Raitakari, O. T. and D. S. Celermajer, Flow-mediated dilatation.    Br J Clin Pharmacol, 2000. 50(5): p. 397-404.-   5. Ter Avest, E., A. F. Stalenhoef, and J. de Graaf, What is the    role of non-invasive measurements of atherosclerosis in individual    cardiovascular risk prediction? Clin Sci (Lond), 2007. 112(10): p.    507-16.-   6. Doshi, S. N., et al., Flow-mediated dilatation following wrist    and upper arm occlusion in humans: the contribution of nitric oxide.    Clin Sci (Lond), 2001. 101(6): p. 629-35.-   7. Duplain, H., et al., Insulin resistance, hyperlipidemia, and    hypertension in mice lacking endothelial nitric oxide synthase.    Circulation, 2001. 104(3): p. 342-5.-   8. Ludmer, P. L., et al., Paradoxical vasoconstriction induced by    acetylcholine in atherosclerotic coronary arteries. N Engl J    Med, 1986. 315(17): p. 1046-51.-   9. Kang, S. M., et al., Relation of vasodilator response of the    brachial artery to inflammatory markers in patients with coronary    artery disease. Echocardiography, 2002. 19(8): p. 661-7.-   10. Kostner, K. M., et al., Inflammation, complement activation and    endothelial function in stable and unstable coronary artery disease.    Clin Chim Acta, 2006. 365(1-2): p. 129-34.-   11. Fronek, A., D. G. DiTomasso, and M. Allison, Noninvasive    assessment of endothelial activity in patients with peripheral    arterial disease and cardiovascular risk factors. Endothelium, 2007.    14(4-5): p. 199-205.-   12. Silvestro, A., et al., Inflammatory status and endothelial    function in asymptomatic and symptomatic peripheral arterial    disease. Vasc Med, 2003. 8(4): p. 225-32.-   13. Chobanian, A. V., Clinical practice. Isolated systolic    hypertension in the elderly. N Engl J Med, 2007. 357(8): p. 789-96.-   14. Sugawara, J., et al., Effect of systemic nitric oxide synthase    inhibition on arterial stiffness in humans. Hypertens Res, 2007.    30(5): p. 411-5.-   15. Yetkin, E., et al., Decreased endothelium-dependent    vasodilatation in patients with migraine: a new aspect to vascular    pathophysiology of migraine. Coron Artery Dis, 2006. 17(1): p.    29-33.-   16. Yetkin, E., et al., Increased dilator response to nitrate and    decreased flow-mediated dilatation in migraineurs. Headache, 2007.    47(1): p. 104-10.-   17. Philip, I., et al., G894T polymorphism in the endothelial nitric    oxide synthase gene is associated with an enhanced vascular    responsiveness to phenylephrine. Circulation, 1999. 99(24): p.    3096-8.-   18. Hingorani, A. D., et al., A common variant of the endothelial    nitric oxide synthase (Glu298-->Asp) is a major risk factor for    coronary artery disease in the UK. Circulation, 1999. 100(14): p.    1515-20.-   19. Lembo, G., et al., A common variant of endothelial nitric oxide    synthase (Glu298Asp) is an independent risk factor for carotid    atherosclerosis. Stroke, 2001. 32(3): p. 735-40.-   20. Rose, K. M., et al., Migraine and other headaches: associations    with Rose angina and coronary heart disease. Neurology, 2004.    63(12): p. 2233-9.-   21. Borroni, B., et al., Endothelial nitric oxide synthase    (Glu298Asp) polymorphism is an independent risk factor for migraine    with aura. Headache, 2006. 46(10): p. 1575-9.-   22. Giugliano, F., et al., Erectile dysfunction associates with    endothelial dysfunction and raised proinflammatory cytokine levels    in obese men. J Endocrinol Invest, 2004. 27(7): p. 665-9.-   23. Cederholm, A. and J. Frostegard, Annexin A5 as a novel player in    prevention of atherothrombosis in SLE and in the general population.    Ann N Y Acad Sci, 2007. 1108: p. 96-103.-   24. Thiagarajan, P. and C. R. Benedict, Inhibition of arterial    thrombosis by recombinant annexin V in a rabbit carotid artery    injury model. Circulation, 1997. 96(7): p. 2339-47.-   25. Rand, J. H., Antiphospholipid antibody-mediated disruption of    the annexin-V antithrombotic shield: a thrombogenic mechanism for    the antiphospholipid syndrome. J Autoimmun, 2000. 15(2): p. 107-11.-   26. Cederholm, A., et al., Decreased binding of annexin v to    endothelial cells: a potential mechanism in atherothrombosis of    patients with systemic lupus erythematosus. Arterioscler Thromb Vasc    Biol, 2005. 25(1): p. 198-203.-   27. Teoh, N. C., et al., Diannexin, a novel annexin V homodimer,    provides prolonged protection against hepatic ischemia-reperfusion    injury in mice. Gastroenterology, 2007. 133(2): p. 632-46.-   28. Shen, X. D., et al., Diannexin, a novel annexin V homodimer,    protects rat liver transplants against cold ischemia-reperfusion    injury. Am J Transplant, 2007. 7(11): p. 2463-71.-   29. Vermes, I., et al., A novel assay for apoptosis. Flow cytometric    detection of phosphatidylserine expression on early apoptotic cells    using fluorescein labelled Annexin V. J Immunol Methods, 1995.    184(1): p. 39-51.

1. A method for the treatment of vascular dysfunction comprisingadministering a therapeutically effective amount of Annexin A5 or afunctional analogue or variant thereof to a patient in need of suchtreatment, wherein the functional analogue or variant of Annexin A5comprises a protein which is more than 90% identical to human AnnexinA5, SEQ ID NO:1.
 2. The method according to claim 1 wherein the vasculardysfunction is associated with impaired endothelium mediatedvasodilatation, a reduced endothelial nitric oxide synthase (eNOS)activity, and/or a reduced nitric oxide (NO) bioavailability.
 3. Amethod for reducing ischemic pain by administering a therapeuticallyeffective amount of Annexin A5 or a functional analogue or variantthereof to a patient in need of such treatment, wherein the functionalanalogue or variant of Annexin A5 comprises a protein which is more than90% identical to human Annexin A5, SEQ ID NO:
 1. 4. The method accordingto claim 1, wherein the treatment reduces the risk of the onset of acutemyocardial infarction (AMI).
 5. The method according to claim 1, whereinthe patient is suffering from a disease selected from angina pectoris,ischaemic heart disease, peripheral artery disease, systolichypertension, migraine, type 2 diabetes and erectile dysfunction.
 6. Themethod according to claim 1, wherein the Annexin A5 or the functionalanalogue or variant thereof is selected from: a) human Annexin A5 (SEQID NO:1), b) a mammalian orthologue of human Annexin A5, c) an allelicor genetic variant of a) or b), d) a functional analogue of Annexinwhich is a protein which is more than 75%, such as more than 80%, morethan 90%, or even more preferably more than 95% identical to humanAnnexin A5, SEQ ID NO:1, e) a dimer of, or fusion protein comprising,any of a), b), c) or d), and f) a PEGylated variant of any of a), b),c), d) or e).
 7. The method according to claim 1, wherein thetherapeutically effective amount of Annexin A5 or a functional analogueor variant thereof is administered parenterally, intravenously,intra-arterially, intra-peritoneally, intra-muscularly orsubcutaneously.
 8. A method for the treatment or reduction of risk ofonset of a vascular disease that is associated with impaired endotheliummediated vasodilatation, a reduced eNOS activity, and/or a reduced NObioavailability comprising administering a therapeutically effectiveamount of Annexin A5 or a functional analogue or variant thereof to apatient in need of such treatment, wherein the functional analogue orvariant of Annexin A5 comprises a protein which is more than 90%identical to human Annexin A5, SEQ ID NO:1.
 9. The method according toclaim 8 wherein the treatment reduces the risk of the onset of acutemyocardial infarction (AMI).
 10. The method according to claim 8,wherein the vascular disease is selected from angina pectoris, ischaemicheart disease, peripheral artery disease, systolic hypertension,migraine, type 2 diabetes and erectile dysfunction.
 11. The methodaccording to claim 8, wherein the Annexin A5 or the functional analogueor variant thereof is administered in conjunction with a thrombolytictherapeutic such as tissue plasminogen activator, urokinase, or abacterial enzyme.
 12. The method according to claim 8, wherein theAnnexin A5 or the functional analogue or variant thereof is administeredin conjunction with an anti-platelet agent such as clopidogrel oraspirin.
 13. The method according to claim 8, wherein the Annexin A5 orthe functional analogue or variant thereof is selected from; a) humanAnnexin A5 (SEQ ID NO:1), b) a mammalian orthologue of human Annexin A5,c) an allelic or genetic variant of a) or b), d) a functional analogueof Annexin which is a protein which is more than 95% identical to humanAnnexin A5, SEQ ID NO:1, e) a dimer of, or fusion protein comprising,any of a), b), c) or d), and f) a PEGylated variant of any of a), b),c), d) or e).
 14. The method according to claim 8, wherein thetherapeutically effective amount of Annexin A5 or a functional analogueor variant thereof is administered parenterally, intravenously,intra-arterially, intra-peritoneally, intra-muscularly orsubcutaneously. 15-28. (canceled)
 29. The method according to claim 3wherein the treatment reduces the risk of the onset of acute myocardialinfarction (AMI).
 30. The method according to claim 3 wherein thepatient is suffering from a disease selected from angina pectoris,ischaemic heart disease, peripheral artery disease, systolichypertension, migraine, type 2 diabetes and erectile dysfunction. 31.The method according to claim 3 wherein the Annexin A5 or the functionalanalogue or variant thereof is selected from; a) human Annexin A5 (SEQID NO:1), b) a mammalian orthologue of human Annexin A5, c) an allelicor genetic variant of a) or b), d) a functional analogue of Annexin A5which is a protein which is more than 95% identical to human Annexin A5,SEQ ID NO:1, e) a dimer of, or fusion protein comprising, any of a), b),c) or d), and f) a PEGylated variant of any of a), b), c), d) or e). 32.The method according to claim 3 wherein the therapeutically effectiveamount of Annexin A5 or a functional analogue or variant thereof isadministered parenterally, intravenously, intra-arterially,intra-peritoneally, intra-muscularly or subcutaneously.
 33. Apharmaceutical composition comprising a therapeutically effective amountof Annexin A5 or a functional analogue or variant thereof for thetreatment of a vascular disease selected from angina pectoris, ischaemicheart disease, peripheral artery disease, systolic hypertension,migraine, type 2 diabetes and erectile dysfunction, wherein thefunctional analogue or variant of Annexin A5 comprises a protein whichis more than 90% identical to human Annexin A5, SEQ ID NO:1.
 34. Thepharmaceutical composition according to claim 33 wherein the Annexin A5or the functional analogue or variant thereof is selected from; a) humanAnnexin A5 (SEQ ID NO:1), b) a mammalian orthologue of human Annexin A5,c) an allelic or genetic variant of a) or b), d) a functional analogueof Annexin A5 which is a protein which is more than 95% identical tohuman Annexin A5, SEQ ID NO:1, e) a dimer of, or fusion proteincomprising, any of a), b), c) or d), and f) a PEGylated variant of anyof a), b), c), d) or e).
 35. The pharmaceutical composition according toclaim 34 intended for parenteral, intravenous, intra-arterial,intra-peritoneal, intra-muscular or subcutaneous administration.